Glucosinolates estimation from de-oiled rapeseed cake using a spectrophotometer

Introduction

Glucosinolates are plant secondary metabolites. Myrosinase is an enzyme that catalyzes the breakdown of glucosinolates. Both of them are usually present in different cells in the tissue, unless there is damage. The breakdown products differ depending on the environmental conditions.

Applications

1. Total glucosinolates estimation from various plants. This protocol aims at the mustard de-oiled cake.

Advantages

1. Less laborious than HPLC
2. Simple extraction and estimation
3. Relatively low cost

Limitations

1. This method has different affinities for different types of glucosinolates. This might lead to a skewed estimation of total Glucosinolate content. For example, sodium tetrachloropalladate reacts to all types of glucosinolates but has a higher affinity to indolyl glucosinolates.

Reagents

2 mM sodium tetrachloropalladate:

58.8 mg Sodium tetrachloropalladate + 170 µl concentrated HCl +100 ml double distilled water.

Procedure

The protocol can be divided into extraction and estimation.

Extraction of total Glucosinolates

1. Homogenise 0.1 g defatted seed meal in a 2 ml vial with 80% methanol.
2. Keep overnight at room temperature.
3. Spin it at 3000 rpm for 4 min
4. Collect supernatant.
5. Make the volume to 2 ml with 80% methanol.

Estimation of total Glucosinolates

1. Take 100µL of the solution from the extraction step.
2. Add 0.3 ml double distilled water
3. 3 ml of 2 mM sodium tetrachloropalladate
4. incubation at room temperature for 1 h
5. The reaction mix turns brown. The intensity of the browning depends on the amount of glucosinolates.
6. A blank was set following the same procedure without the extract.
7. Find the O.D at 425nm use the O.D in the following formula to get the quantity of total Glucosinolates in µmol/g (Y).
8. Y = 1.40 + 118.86 × A425
9. Total glucosinolate content obtained by the predicted formula was comparable with the HPLC results.

Caution

• Using the right volumes is the key to the success of this protocol.
• The extract preparation is very crucial, and therefore utmost care is needed before quantification, such as avoiding excessive shaking and vortexing of the extract. A brief centrifuge would suffice for good results.

Reference

1. A simple spectrophotometric method for estimating total glucosinolates in mustard de-oiled cake.
2. Quantitative analysis of total glucosinolate content in concentrated extracts from double low rapeseed by the Pd-glucosinolate complex method.

3. Small variation of glucosinolate composition in Japanese cultivars of radish (Raphanus sativus L.) requires simple quantitative analysis for breeding of glucosinolate component.